Aedes albopictus cell line. The potential of these viruses to cause severe disease at primary infection is of interest although few studies have been performed with these two Dengue serotypes.Proteomic analysis was performed to identify proteins regulated during infection by Dengue serotypes 1 and 3 in an translation of some host genes, which may result in stress due to responses involving unfolded proteins.The most relevant observation of our study is the significant overexpression of proteins involved in the cellular stress response and the glycolysis pathway after 48 hours of infection. Viral infection activates the Therefore, the oxidation reduction and glycolytic mechanisms could participate in the antiviral response against Dengue virus. The results of our study should help to improve our knowledge of the virus-mosquito interaction at a cellular level with the aim of designing efficient strategies for the control of Dengue virus. Aedes aegypti  is considered the major vector for Dengue infection outbreaks worldwide [Aedes albopictus  is a less efficient vector for this virus, although it was involved in Dengue outbreaks in Japan, Seychelles, Hawaii, and Reunion Island [orldwide . Aedes an Island . The recn Island ,4. Dengun Island . The Worn Island . DENV drn Island ,7 as a cn Island .There are four serotypes of DENV (DENV-1 to DENV-4) that differ by their antigenic groups. Each group can be divided into three to five different genotypes. Therefore, it is difficult to take into consideration all these factors when trying to determine the mechanisms involved in the pathogenesis of the virus. Many researches have focused on DENV-2, which provokes the most severe form of Dengue in secondary infection ,9, but tAe. albopictus cell line using two-dimensional differential in-gel electrophoresis (2D-DIGE) to examine modulated proteins of infected mosquito cells. We chose DENV-1 and DENV-3 as study models because of their remarkable capacities to induce severe disease with the first infection. Furthermore, travelers have more chance to develop DENV-1 or DENV-3 infection when they live in non-endemic areas and have never been in contact with the virus [Because there is no available antiviral treatment or vaccine to cure or prevent DENV, other approaches are needed to fight and control the virus. A good understanding at the molecular level of the virus-mosquito interaction should help with the design of efficient strategies for the control of DENV. Mosquito cell infection is a part of the Dengue viruses' life cycle that is poorly understood. In this study, we compared the proteome of infected and non-infected cultures of the C6/36 phoresis D-DIGE tohe virus ,15. Thishe virus .C6/36 cells were collected 48 h after infection with DENV-1, DENV-3, or a control (hereafter "mock infected"). We chose to use 48 h post infection because it was previously shown that this time corresponded to the exponential phase for both DENV-1 and DENV-3 productions in C6/36 cells . We assuTwenty two spots were identified as being 13 different proteins by Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) Table . SeveralNine of the 13 identified proteins belong to the groups 1 and 2, i.e., those proteins that are up-regulated in infected conditions. Notably, five of these were enzymes involved in the glycolysis pathway. These included enolase, phosphoglycerate mutase, triosephosphate isomerase, fructose-bisphosphate aldolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPD). The significant up-regulation of these enzymes in virus-infected cells might indicate that the virus disturbs glucose metabolism in host cells . The fouThe four overexpressed proteins in mock-infected cells were procollagen-lysine 2-oxyglutarate 5-dioxygenase (PLOD), calponin, ethanolamine-phosphate cytidylyltransferase, and aconitase. PLOD is involved in the oxidation-reduction necessary for the biosynthesis of collagen ; calponiAe. albopictus cells infected and non-infected with DENV-1 and DENV-3.DENV usually induces apoptosis in infected mammalian cells but causes less damage in mosquito cells . The mecAe. albopictus cells infected with Mayaro virus, and thus protect the cells from injury during viral infection [Cells infected with DENV-1 and DENV-3 exhibited many proteins that were differentially expressed compared to the mock-infected cells. These data may reveal cellular stress due to viral infection. In response to this stress, mosquito cells utilize antioxidant reactions combined with increased energy production to cope with the virus. The up-regulation of chaperone proteins in infected cells might be due to stress in the endoplasmic reticulum (ER) induced by viral infection; ER is where proteins are synthesized. Only proteins that are correctly folded by ER chaperones are transported to the Golgi apparatus. Misfolded and unfolded proteins lead to ER stress. Eukaryotic cells up-regulate ER chaperones and establish the cytoprotective mechanism known as the unfolded protein response (UPR) to degrade the unfolded protein . Moleculnfection . Furthernfection .Culex quinequefasciatus (B0WBF6_CULQU). These proteins belong to the heat-shock protein families Hsp60 and Hsp70, respectively.Interestingly, it has been reported that acute infection of mammalian cells with several types of viruses often results in the induction of heat-shock protein expression . The up-Cells require a chaperone function to prevent or correct misfolded or unfolded proteins created by environmental stress. These chaperone proteins aid protein folding in different ways. Hsp60 is a mitochondrial chaperone that generally assists the carrying and refolding of proteins from the cytosol to mitochondria . Many Hs2+-binding chaperone protein [The modulation of chaperone-associated proteins could protect cells from apoptosis, as does the overexpression of calreticulin, which is a Ca protein . These f protein ,29. In t protein .+, reduces ubiquinone, and transports protons across the inner mitochondrial membrane. This enzyme complex also reduces O2 to superoxide, which causes cellular oxidative stress [Chen et al.  further e stress . This meTrypanosoma; this finding could represent a new drug target against parasites [via their vector control. The increase in glycolytic enzymes could also be a result of the arrest at the G1 cell cycle due to UPR.Another interesting result of our study is that five enzymes involved in the glycolysis pathway were up-regulated. Increased glucose uptake and glycolytic enzyme activity due to viral infection have been found in other studies ,40. The arasites ,43. In aarasites . These ein vivo in mosquito tissues. Improvements in our knowledge on mosquito cell systems will be important to decipher the infection process of dengue virus in human.The modulation of protein expression found in our study might be the strategy of the virus to overcome host pathways to facilitate survival at the expense of the host. Further studies are needed to understand the mechanism by which these proteins are induced during viral infection. Virus could stimulate the transcription and translation of some host products for their survival, but the mechanism is still unknown. The modulation of protein expression could also be the mosquito's response to the viral infection. It would be interesting in a further study, to determine whether a similar response occurs in cells infected with another virus or if the observed modulation of protein expression is specifically induce in response to DENV infection. Mosquitoes generate oxido-reduction stress to cope with the virus and stimulate the glycolysis pathway to prevent cellular damage. Thus, it might be beneficial to understand the proteome of infected cells to develop an anti-pathogen approach. However, these proteomics studies need to be complemented by studies using RNAi gene silencing to allow the characterization of modulated genes Ae. Albopictus C6/36 cells were grown in minimal essential medium supplemented with 10% fetal calf serum, 1% L-glutamine, 1% sodium bicarbonate, 1% nonessential amino acids, 50 U/mL of penicillin, and 50 μg/mL of streptomycin at 28°C [Ae. albopictus cells. at 28°C . Cells w at 28°C . Mock-inInfected or mock-infected cells were washed with PBS and then lysed by solubilizing buffer . After a centrifugation at 16000 × g for 45 min, the supernatants were collected and protein concentrations were measured using a 2-D Quant kit .For analytical 2D-DIGE, DENV-1 and DENV-3 infected and non-infected protein samples were compared using the CyDye DIGE Fluors for Ettan DIGE . Proteins were labelled according to the Ettan DIGE minimal labelling protocol . For each sample , 50 μg of protein was labeled with 400 pmol of either Cy3 or Cy5. The internal standard was a pool of equal amounts (25 μg) from all samples which was labeled with Cy2. These labeled samples were then combined and loaded on gels. Every gels contained 50 μg of sample labeled with Cy3, 50 μg of sample labeled with Cy5 and 50 μg of internal standard labeled with Cy2. The sample volume was made up to 450 μL by adding rehydration buffer  prior to separation by isoelectric focusing (IEF). IEF was performed with 24 cm Immobiline DryStrip, pH 3-10 NL. The run conditions were as follows: rehydration for 14 h at 20°C, current of 50 μA per strip, 60 V (step) for 3 h, 1000 V (gradient) for 4 h, 8000 V (gradient) for 4 h, and 8000 V (step) until reaching a total of 64 000 Vh. 2-DE was performed on 12% SDS-PAGE gel at 15 mA/gel for 6 h and then at 30 mA/gel until the bromophenol blue front reached the end of the gel. Gels were scanned using a Typhoon 9400 imager (Amersham Biosciences). All gel images were acquired at 100 μm pixel resolution under nonsaturating conditions. 2D-DIGE images were analyzed using Progenesis SameSpots 3.1 software. Statistical analysis and protein quantification were carried out using this software with ANOVA which took into account the mean difference and the variance among 3 groups: DENV-1, DENV-3 and mock-infected groups. The fold change with a cut-off of 2.0-fold up- or down-regulated was used . The statistical power of this study was greater than 0.8. Protein spots with a significant altered expression (p < 0.001) were trypsin digested and identified with mass spectrometry.http://www.expasy.ch and by using the MASCOT v 2.2 algorithm (http://www.matrixscience.com) with trypsin enzyme specificity and one trypsin missed cleavage allowed [For 2D-DIGE, gels were run with 150 μg of a mix of protein from the different samples  and these gels were afterwards stained with CBB. Spots of interest were localized on the gels by comparing the CBB-stained spot pattern with the 2D-DIGE protein pattern. To ensure consistency, gels were analysed using Progenesis SameSpots 3.1 software and differential spots were identified. Gel image containing differential spots and picking gels were compared physically side-by-side and the candidate spots were excised manually in a laminar flow hood. Enzymatic in-gel digestion through peptide spotting and protein identification was performed as previously described ,47. Brie allowed . CarbamiAedes: Ae; Japanese encephalitis virus: JEV; DHF: Dengue Hemorrhagic Fever; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; UPR: Unfolded protein response; PLOD: Procollagen-lysine 2-oxyglutarate 5-dioxygenase; HSP: Heat shock protein; MALDI-TOF MS: Matrix assisted laser desorption ionisation time-of-flight mass spectrometry.Dengue Virus: DENV; The authors declare that they have no competing interests.Conceived and designed the experiments: SP, DM. Performed the experiments: SP, PS, MS. Analyzed the data: SP, PS, DM. Wrote the paper: SP, DM. Corrected the manuscript: VC, FT. All authors read approved the final version of the MS


The increased risk of cardiovascular disease (CVD) in diabetic compared to non-diabetic subjects seems to decrease with age. Whether this age-related reduction applies to CVD risk factors, and whether it is limited to established diabetes mellitus (DM) or also applies to pre-diabetic conditions are not well known.Using a cross-sectional design we compared the strength of the correlation between glucometabolic disturbances (by grouping), CVD risk factor burden and self-rated health, in two age groups: middle-aged (57-69 years) and older (70-86 years) subjects, (63% men), participating in the Malmö Preventive Project Re-examination Study . Simple (unadjusted) logistic regression analysis was applied to estimate between-group differences and trends. Interaction analysis was applied to estimate differences between age groups.CVD risk factor burden and the proportion of subjects reporting poor self-rated health increased with increasing glucometabolic disturbance for men and women in both age groups . The slope of the trend curve with increasing CVD risk factor burden was significantly steeper for older women than for older men (p-interaction = 0.002). The slope of the trend curve for poor self-rated health was significantly steeper for middle-aged than for older men (p-interaction = 0.005), while no difference was observed between the age groups among women (p-interaction = 0.97).We found no reduction in risk factor accumulation with increasing glucometabolic disturbance between middle-aged and older subjects. Our results indicate life-long CVD risk factor clustering with increased glucometabolic disturbance, and suggest that previously observed age-related reduction in excess CVD risk for subjects with DM might be due to a survival bias. However, our observations indicate more pronounced risk factor clustering and worse self-rated health with increased glucometabolic disturbance in older women than in older men. Diabetes mellitus (DM) is a well-established risk factor for cardiovascular disease (CVD). The Framingham Study was the first to describe a 2-3 fold increase in the risk of CVD in diabetic compared to non-diabetic subjects, with women bearing a disproportionate burden of the risk . FastingThe incidences of glucometabolic disturbances and CVD increase with age. However, it has been shown that the increased incidence of CVD in diabetic compared to non-diabetic subjects seems to decrease with increasing age . WhetherThe Malmö Preventive Project was a population-based cohort study conducted between 1974 and 1992. The project was a preventive case-finding program with the aim of screening for CVD risk factors, alcohol abuse and breast cancer in the population . Birth c1c were measured at a separate visit [The Malmö Preventive Project Re-examination Study was conducted at Skåne University Hospital in Malmö during the period 2002-2006. The target population consisted of the approximately 25,000 individuals in the original Malmö Preventive Project cohort, still alive and living in the Malmö area. In total, 18,238 subjects participated (63% men) giving a participation rate of approximately 72%. At the first visit the subjects were given verbal information on the study, and blood samples were drawn (after overnight fasting). Laboratory tests included FPG, serum (s-) total cholesterol, s-triglycerides and s-high-density lipoprotein . Low-density lipoprotein cholesterol was calculated using the Friedewald formula [te visit .Data quality was high, with missing values ranging from 0.1% (laboratory measures) to 0.5%  to 1.5% (questionnaire data). All participants received information on the results of their laboratory tests and blood pressure measurements, and were offered an appointment with a physician in cases of new-onset type-2 DM (T2DM), dyslipidemia or hypertension, if they did not have a personal family physician.The Ethics Committee of Lund University, Sweden, approved the Malmö Preventive Project Re-examination Study (No. LU 244-02). The study complied with the Declaration of Helsinki. All participants signed an informed consent form before entering the study.th group constituted subjects with impaired fasting glucose (IFG) according to the World Health Organization (WHO) criteria (FPG 6.1-6.9 mmol/L) [th subjects with new-onset T2DM, and the 6th group patients with established DM (T1DM or T2DM). DM was classified as established if self-reported in the questionnaire (T1DM or T2DM) and/or if the subject was taking prescription drugs for DM. New-onset T2DM was defined by two separate FPG values ≥ 7.0 mmol/L [th group. This was done to maximize the benefit of having two separate measurements and thus minimize the risk of mis-classification.In the current study, the subjects were divided into six groups according to glucometabolic status. In the first three groups the FPG was within the normal range . The 4 mmol/L) , the 5th0 mmol/L . AdditioFive CVD risk factors were chosen for this study: the presence of uncontrolled hypertension, dyslipidemia, central obesity, current smoking and lack of physical activity. Uncontrolled hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg, irrespective of hypertension treatment. Dyslipidemia was defined as at least one of the following: total cholesterol ≥ 5.0 mmol/L, low-density lipoprotein ≥ 3.0, triglycerides ≥ 1.7 mmol/L and/or high-density lipoprotein < 1.0 mmol/L for men and < 1.3 mmol/L for women. Central obesity was defined as a waist circumference of ≥ 102 cm for men and ≥ 88 cm for women. These limits were based on the European Society of Cardiology guidelines for CVD prevention  and classification of the metabolic syndrome  ,12. Currst to the 6th group for SRH and from the 1st to the 5th group for the risk factors. The 6th group (established DM) was excluded in the latter case as non-linearity could be expected, as the result of better diagnosis and treatment of risk factors for subjects with established DM. Ratios between the β-coefficients obtained from the trend analyses were then calculated, and interaction analysis was performed to estimate differences between the age groups, by entering variable A *variable B into the regression analysis. A traditional double-sided significance level of p < 0.05 was used. Baseline characteristics are presented as means ± standard deviations and percentages. The SPSS 19.0 computer package was used for statistical analysis .All calculations were stratified by gender and age: middle-aged (57-69 years) and older (70-86 years). The proportions (%) of subjects having at least three of the five selected risk factors and low SRH were calculated for each of the six glucometabolic status groups described above. Differences in proportions between age groups within each glucometabolic status group were calculated by using simple (unadjusted) logistic regression, generating odds ratios (OR) and 95% confidence intervals (95% CI). To assess the strength of the correlation between increasing glucometabolic disturbance and increased risk factor burden and worse SRH, logistic regression analysis was used to calculate beta (β)-coefficients for trends from the 1The total Malmö Preventive Project Re-examination Study cohort n = 18,238) was used in this study. Twelve non-diabetic subjects lacked FPG values and could thus not be categorized. Baseline characteristics for men and women are given in Table 8,238 wasst through the 5th glucometabolic group in both age groups (p-trend < 0.0001). The ratio between the β-coefficients from the trend tests (reflecting the slope of the curve) for middle-aged vs. older men was 1.21 (p-interaction = 0.12) and for women 0.97 (p-interaction = 0.78). Although the difference in trends between age groups was not significant among men, older subjects with FPG = 5.1-5.5 mmol/L (OR 0.78 (95% CI 0.67-0.92) p = 0.002), IFG (0.80 (0.67-0.96), p = 0.02), new-onset T2DM (0.37 (0.22-0.65), p = 0.0004) and established DM (0.78 (0.62-0.98), p = 0.03) had significantly fewer risk factors than the middle-aged subjects in the respective groups. No such differences between age groups were seen among women. The ratio between β-coefficients for middle-aged women vs. middle-aged men was 1.19 (p-interaction = 0.11) while for older women vs. older men it was 1.49 (p-interaction = 0.002).The proportions (%) of subjects within each glucometabolic status group and age group having at least three of the five selected risk factors are shown in Figures th group) reported significantly better SRH than older men in these groups, while there was no difference in SRH between middle-aged and older men with new-onset and established DM. Consequently, the trend from the 1st to the 6th groups for the middle-aged men was stronger  than for the older men , giving a ratio of 1.89 (p-interaction = 0.005). For women, the β-coefficient for middle-aged women was 0.17 (p-trend < 0.0001) compared to 0.14 (p-trend < 0.0001) for older women; the ratio being 1.22 (p-interaction = 0.47). The differences in β-coefficients between men and women in each age group were not statistically significant .The proportions (%) of subjects within each glucose group and age group reporting low SRH are presented in Figures In this population-based cohort study of middle-aged and older subjects, we observed that CVD risk factor burden increased linearly with increasing glucometabolic disturbance among men and women in both age groups. The slope of the trend curve was not significantly steeper for middle-aged than older subjects of either sex, indicating that there was no significant attenuation of the relation between increasing glucometabolic disturbance and CVD risk factor burden with age. However, older men with IFG, new-onset DM and established DM had significantly fewer risk factors than middle-aged men in the corresponding glucometabolic status groups; no such difference was observed among women. Consequently, the slope of the trend curve was significantly steeper for older women than older men, indicating a higher risk factor accumulation with increasing glucometabolic disturbance among the older women. We observed that older subjects of both genders reported worse SRH than middle-aged subjects. This difference decreased with increasing glucometabolic disturbance among men, but not among women.Although the excess CVD risk due to DM in women compared to men seems to have declined since first described by the Framingham investigators in the 1970s, recent studies still show that women bear a disproportionate part of the DM-related excess risk ,15. TherWe have not been able to find any studies reporting age- and gender-stratified CVD risk ratios or risk factor burdens across the spectrum of glucometabolic disturbances. Several studies on CVD risk ratios between diabetic and non-diabetic subjects have, however, been reported, some of which have presented gender-specific, age-stratified results. A Norwegian study on coronary heart disease mortality showed a decline in risk ratio from the youngest (< 60 years) to the oldest (> 80 years) age groups, equally so for men and women . In the In our study, the risk factor burden increased significantly across the glucometabolic status groups for men and women in both age groups. Although there were significant differences between middle-aged and older men with IFG, new-onset and established DM as discussed above, the difference in trends for risk factor accumulation across glucometabolic status groups between the two age groups was not significant for either sex. This discrepancy might reflect a survival bias, in that subjects with glucometabolic disturbances experience a life-long tendency for risk factor clustering while those with the most serious metabolic disturbances succumb to CVD events at an earlier age. However, it should be noted that the trend was significantly weaker among older men than older women, who showed an accumulation of risk factors with increasing glucometabolic disturbance to the same degree as both middle-aged women and men. These results support the fact that not only women with DM but also prediabetic women, irrespective of age, need special attention when screening for and treating concomitant CVD risk factors.The major strength of this study is that it examines the association between CVD risk factor profile and degree of glucometabolic disturbance through the range from NFG to IFG and DM, in a large population-based cohort, allowing comparison between different groups and the assessment of trends and interactions. The participation rate in the study was high (72%) and missing values were few, increasing the validity of the results.There are some limitations in this study. Firstly, as with all population-based cohort studies, there is risk of a "healthy cohort" effect, meaning that subjects with co-morbidities, disabilities or poor quality of life might either have died or been too weak to participate in the study. As these subjects would be more likely to be older and classified in the groups of subjects with higher glucometabolic disturbances, such a bias would lead to underestimation of CVD risk factor burden and poor SRH in those groups. A study on attendees versus non-attendees was performed in the original Malmö Preventive Project, showing that total and cause-specific mortality was higher in non-participants than in those participating in the study . It can In conclusion, the previously observed age-related reduction in elevated risk of CVD in diabetic compared to non-diabetic subjects was not observed for risk factor accumulation with increasing glucometabolic disturbance. Our results indicate life-long risk factor clustering with increasing glucometabolic disturbance, while an age-related CVD risk attenuation with respect to DM might be due to a survival bias. However, our observations suggest a more pronounced risk factor clustering and a more negative effect on SRH with increasing glucometabolic disturbance in older women than in older men. These results may contribute to the previously observed higher risk of CVD in women with glucometabolic disturbances than in men. The results also indicate the need to pay special attention to screening for and treating concomitant CVD risk factors among women with glucometabolic disturbances, irrespective of age.CVD: cardiovascular disease; DM: diabetes mellitus; FPG: fasting plasma glucose; IFG: impaired fasting glucose; SRH: self-rated health; T1DM: Type-1 diabetes mellitus; T2DM: Type-2 diabetes mellitus.The authors declare that they have no competing interests.ML designed the study in collaboration with PN and RW. ML carried out the statistical analysis and drafted the manuscript. MP was in charge of data collection in the Malmö Preventive Project Re-examination Study. RW, MP and PN critically revised the manuscript for intellectual content. All authors read and approved the final manuscript.


There is recent interest surrounding the use of the triglyceride-to-HDL cholesterol ratio as a surrogate marker of insulin resistance in clinical practice, as it may identify people at high risk of developing diabetes or its complications. However, it has been suggested using this lipid ratio may not be appropriate for measuring insulin resistance in African-Americans, particularly women. We investigated if this inconsistency extended to South Asian women in a UK multi-ethnic cohort of White Europeans and South Asians.Cross-sectional analysis was done of 729 participants from the ADDITION-Leicester study from 2005 to 2009. The association between tertiles of triglyceride-to-HDL cholesterol ratio to fasting insulin, homeostatic model of assessment for insulin resistance (HOMA1-IR), quantitative insulin sensitivity check index (QUICKI) and glucose: insulin ratio was examined with adjustment for confounding variables.th percentile (3.08), was 0.74 (0.67 to 0.81), 0.72 (0.65 to 0.79), 0.75 (0.66 to 0.85) and 0.67 (0.56 to 0.78) in White European men and women, South Asian men and women respectively. The optimal cut-points for detecting insulin resistance were 0.9–1.7 in mmol/l (2.0–3.8 in mg/dl) for the triglyceride-to-HDL ratio.Incremental tertiles of the triglyceride-to-HDL cholesterol ratio demonstrated a significant positive association with levels of fasting insulin, HOMA1-IR, glucose: insulin ratio and a negative association with QUICKI in White European men (n = 255) and women (n = 250) and South Asian men (n = 124) , but not South Asian women (n = 100). A significant interaction was demonstrated between sex and triglyceride-to-HDL cholesterol ratio tertiles in South Asians only (p<0.05). The area under the receiver operating characteristic curve for triglyceride-to-HDL cholesterol ratio to detect insulin resistance, defined as the cohort HOMA1-IR≥75In South Asian women the triglyceride-to-HDL cholesterol ratio was not associated with insulin resistance; therefore there may be limitations in its use as a surrogate marker in this group. The triglyceride-to-HDL cholesterol ratio has been investigated recently for various potential clinical uses in adult and paediatric populations South Asians form over one-fifth of the world's population, including over 4 million migrants living in both the US and UK The analysis was performed using data from the ADDITION-Leicester study . ADDITION-Leicester consisted of a population based screening program to identify individuals with diabetes who then underwent a five year cluster randomised controlled trial. This study contributed to the multi-centre ADDITION-Europe, but was also a stand-alone study designed to investigate differences between westernised South Asians and White Europeans Fasting lipid levels were collected in lithium heparin bottles and measured on a Dade Behring Dimension analyser, Newark, USA. HDL cholesterol was measured after isolation of other non HDL apolipoproteins; triglycerides were measured following enzymatic hydrolysis to glycerol. Glucose samples were collected in fluoride oxalate tubes and measured on an Abbott Aeroset clinical chemistry  analyser. Glucose and lipid profiles were measured on the same day in the same laboratory with quality control assessments conducted daily. Fasting insulin levels were collected in lithium heparin tubes and centrifuged immediately, before stored in a −80 degrees Celsius freezer. Insulin samples were analysed on a Perkin Elmer time-resolved fluoro-immuno assay on an Auto DELFIA, which has less than 1% cross-reactivity with C-peptide and proinsulin. The inter- and intra-coefficient of variations were less than 2.5% for insulin, HDL, triglycerides and glucose.SPSS version 18.0  was used to perform statistical analysis, except that XLSTAT version 2011.5  was used for receiver operating characteristic (ROC) curve analysis. Baseline demographics of the analysed population were reported stratified by ethnic group and sex. The distribution of continuous variables was inspected for any outlying values, skewness and kurtosis. Means and standard deviations were reported for normally distributed variables. Non-normally distributed parameters were logarithmically transformed and results expressed as geometric mean with 95% confidence intervals. Discrete variables were analysed using chi-squared tests. A high triglyceride level was defined as ≥1.7 mmol/l and low HDL level as <1.0 mmol/l and <1.3 mmol/l in men and women respectively th percentile was defined as insulin resistance, following similar use in other studies th percentile (hyperinsulinaemia) in people without diabetes Within each sex and ethnic group, the triglyceride-to-HDL cholesterol ratio was divided into thirds (tertiles) and tested for association with markers of insulin resistance using analysis of covariance modelling with adjustment for confounding variables. Insulin resistance was assessed using the following four markers. Homeostasis model assessment of insulin resistance (HOMA1-IR) was calculated as fasting glucose (mmol/l)×fasting insulin (µIU/ml)/22.5 which correlates well with insulin resistance th percentile (3.08), by calculating the area under the receiver operating characteristic (AUROC) curve and its 95% confidence intervals Finally, we analysed performance of the triglyceride-to-HDL cholesterol ratio to detect insulin resistance, as measured by the cohort HOMA-IR≥75Baseline demographics of the cohort investigated are shown in th percentile and a negative association with QUICKI , 0.71 (0.64 to 0.78), 0.74 (0.62 to 0.83) and 0.68 (0.56 to 0.80) in White European men and women, South Asian men and women respectively . For triThe main findings of this study demonstrate that the triglyceride-to-HDL cholesterol ratio is associated with insulin resistance measures in White Europeans and South Asian men; this relationship appeared to be less established in South Asian women. However the test performance of triglyceride-to-HDL cholesterol ratio to detect insulin resistance was variable, with the 95% confidence intervals of the area under the ROC curve falling below the acceptable range in all four groups tested. Finally the optimal cut-points for triglyceride-to-HDL cholesterol ratio and triglyceride to detect insulin resistance were 0.9 to 1.7 and 1.4 to 1.8 mmol/l respectively.Previous studies have reported the triglyceride-to-HDL cholesterol ratio is a valid marker of insulin resistance, particularly in White populations Despite our novel finding, we acknowledge the number of South Asian women in the present study was limited to 100 and therefore results may be somewhat prone to power issues. The percentage of South Asian women with insulin resistance defined by fasting insulin and HOMA-IR values in the upper quartile appeared to show increasing trend across incremental triglyceride-to-HDL cholesterol ratio tertiles but neither result achieved statistical significance. Therefore our results should be considered as hypothesis generating and investigated further in well-designed prospective studies. Furthermore, other potential confounding factors cannot be accounted for which may have influenced our results. These include physical activity levels and dietary intake, including alcohol consumption. Also, our South Asian population was a mix of first and second generation immigrants. Finally, medications which affect insulin or HDL-cholesterol levels, such as hormone replacement therapy/post-menopausal oestrogens, were not known as only cardiovascular medications were electronically recorded in ADDITION-Leicester databases. However South Asian women were not over-represented in exclusions made due to lipid lowering therapies; also there were no exclusions made due to out-lying insulin values in South Asian women.We acknowledge the HOMA-IR values in the present study may appear to be lower than in some, but not all cohorts investigating the same topic There are a number of strengths of this study. We investigated a multi-ethnic cohort of White Europeans and South Asians, where all participants underwent phenotyping as well insulin measurements. Secondly, we reduced the large intra-individual variation of triglyceride by using fasting samples. Thirdly, the results were stratified by sex and tested for an interaction effect with triglyceride-to-HDL cholesterol ratio. Regarding potential limitations, we were unable to measure insulin resistance directly in this cohort of 729 individuals, a recognised problem in similar studies Our study demonstrated the optimal cut-points for the triglyceride-to-HDL cholesterol ratio to detect insulin resistance were 1.7 and 0.9 in mmol/l international (SI) unit  for White men and women, and 1.2 and 1.1 mmol/l (2.8 and 2.5 mg/dl) for South Asian men and women. Previous studies have demonstrated the same optimal cut-points to detect insulin resistance are between 2.0 to 2.5 mg/dl in African-Americans and 3.0 mg/dl in both non-Hispanic Whites and Mexican-Americans without diabetes. Using international units, a separate study has reported optimal cut-points for detecting insulin resistance of 0.9, 1.1, 1.1 and 1.8 mmol/l for Aboriginal, Chinese, European and South Asian populations. The reported differences between optimal cut-points in various studies may represent a mix of ethnic differences and/or different methods employed, such as measuring insulin resistance directly or using HOMA-IR models.The potential translational value of the triglyceride-to-HDL cholesterol ratio into clinical practice has yet to be determined. Triglycerides and HDL-cholesterol are included within diagnostic criteria of the metabolic syndrome separately. A combined lipid ratio may better reflect the overall interaction between lipid/lipoprotein fractions, and therefore associations with insulin resistance To our knowledge this is the first study to demonstrate the optimal cut-points for triglyceride level detecting insulin resistance in South Asians are lower than those used in the metabolic syndrome, also termed the insulin resistance syndrome (28). International organisations have recommended using lower ethnic specific cut-points for waist circumference and body mass index in the metabolic syndrome for South Asians in response to high levels of diabetes


In soils with a low phosphorus (P) supply, sugar beet is known to intake more P than other species such as maize, wheat, or groundnut. We hypothesized that organic compounds exuded by sugar beet roots solubilize soil P and that this exudation is stimulated by P starvation.Root exudates were collected from plants grown in hydroponics under low- and high-P availability. Exudate components were separated by HPLC, ionized by electrospray, and detected by mass spectrometry in the range of mass-to-charge ratio (m/z) from 100 to 1000. Eight mass spectrometric signals were enhanced at least 5-fold by low P availability at all harvest times. Among these signals, negative ions with an m/z of 137 and 147 were shown to originate from salicylic acid and citramalic acid. The ability of both compounds to mobilize soil P was demonstrated by incubation of pure substances with Oxisol soil fertilized with calcium phosphate.Root exudates of sugar beet contain salicylic acid and citramalic acid, the latter of which has rarely been detected in plants so far. Both metabolites solubilize soil P and their exudation by roots is stimulated by P deficiency. These results provide the first assignment of a biological function to citramalic acid of plant origin. Sugar beet and wheat are similar in their phosphorus (P) efficiency with regard to shoot production  but theyPlants exude up to 30% of assimilated carbon into the rhizosphere . The comP deficiency usually increases the root exudation rate and alters the composition of exudates ,6, whichDifferent mechanisms have been proposed for the facilitation of P acquisition by organic acids. For instance, citrate might increase the availability of P in the soil by binding calcium (Ca) and thus reducing the formation of insoluble complexes of Ca with P . Citrate-1) while the amount of organic acids released by roots is relatively low. Furthermore, organic acids are rapidly decomposed by rhizosphere microorganisms and adsorbed to soil particles. Because the concentration of citrate in the rhizosphere soil solution is very low , the co< 10 μM; ,20. UndeFor plants without cluster roots, i.e., for most plant species, other root exudate compounds, in place of or together with citrate, may play important roles in increasing P availability. For example, the high P-uptake efficiency of pigeon pea is due to the exudation of piscidic acid , which iThe objective of this study was to use HPLC-ESI-MS to identify components of exudates of sugar beet roots that might increase P availability in the soil. Assuming that P-deficient plants would exude larger amounts of the compounds that solubilize soil P, root exudates were collected from sugar beet plants grown in nutrient solution with sufficient and with low P supply. Components of root exudates produced in larger amounts under low P supply were identified by differential metabolic profiling and were tested for their ability to solubilize P in soil.-1. The low P concentration resulted in a decreased yield that was only 10-15% of the yield achieved with the high P concentration  and the same soil-solution ratio (1:5) to compare the ability of organic acids to mobilize P in a Spodic Horizon.Candidate compounds were tested for the ability to solubilize P from soil. Six compounds were selected from the KEGG database  based onx et al.  used the- originated from salicylic acid and that the signal with an m/z value of 147 [M-H]- originated from citramalic acid. Salicylic and citramalic acids were present in root exudates originating from both low- and high-P treatments but concentrations of the two compounds were higher with the low-P than high-P treatment , proved that the root exudate signal with an m/z value of 137 [M-H] Figures .Root exudates were collected from roots grown in nutrient solution which may differ from soil grown roots by, for example, the lack of root hairs. However, dense root hairs are formed in nutrient solution under conditions of P deficiency ; actuallThe ability of salicylic and citramalic acid to mobilize P from soil is related to their carboxyl and hydroxyl functionalities Figure . The effThe effect of salicylic acid on P availability in soil has been studied by Staunton and Leprice , who shoArabidopsis thaliana  and a retention time of 7.15 min on the RP-18 phase. Citramalic acid was identified by the mass transition 147 > 87 [M-H-CH3COOH] and a retention time of 9.2 min on the HILIC phase.HPLC-MS-MS detection of specific compounds was performed on an identical HPLC system with a triple quadrupole mass spectrometer . Chromatography was performed on a polar-modified RP-18 phase and on a HILIC (hydrophilic interaction chromatography) phase. Root exudates and test substances were separated under identical conditions. RP-18 chromatography was conducted as described before . For HIL-1. pH of the solutions was adjusted to 5.6 with NaOH or HCl. Highly P fixing fossile Oxisol, containing mainly P bound to Al and Fe with 788 mg P kg-1 as Fe/Al-P and 330 mg P kg-1 as Ca-P, clay content 50%, pH(CaCl2) 5.6, P concentration in soil solution 0.17 μmol L-1 and Ca acetate-lactate (CAL) extractable P of 4.3 mg kg-1 was fertilized with 100 mg P kg-1 as Ca(H2PO4)2.H2O and moistured to 22% w/w water content. After 10 days of equilibration, 9.76 g of moist soil  was mixed with 40 mL of the solutions of the compounds listed above or with distilled water. Microbial degradation was prevented by adding two drops of toluene. Each treatment was replicated three times. The samples were shaken on a reciprocal shaker at 150 cycles per minute for 5 h, centrifuged for 15 min at the relative centrifugation force of 2660 g, and the supernatant was filtered though a 0.45-μm nylon membrane filter. Inorganic P concentration was determined by a molybdenum blue colorimetric method [Solutions of 4-hydroxy benzoic acid , urocanic acid , salicylic acid , citramalic acid , D-arabino-1,4-lactone , and pantoic acid  were prepared in water at the concentration of 1 mmol Lc method .RK performed hydroponic experiments and P solubilization tests. UH and AR performed metabolic profiling and analysis of root exudate components. BS designed and supervised the P solubilization experiment. PK guided metabolic profiling and wrote scripts for data processing. NC conceived the study. All authors evaluated the data, wrote parts of the manuscript and approved the manuscript for submission.


Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods.Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants,  Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host" in vitro immune response to probiotics is both species- and strain-specific Dendritic cells (DCs) are professional antigen-presenting cells and are essential mediators of immunity and tolerance Lactobacillus paracasei CNCM I-4034 In a previous study, a novel LAB strain was isolated from the faeces of exclusively breast-fed newborn infants and selected based on its probiotic properties, such as adhesion to intestinal mucus, sensitivity to antibiotics and resistance to gastrointestinal juices, biliary salts, NaCl and low pH. We identified this strain as L. paracasei CNCM I-4034 and its cell-free culture supernatant (CFS) to activate human intestinal DCs, to determine how they respond to pathogenic bacteria, specifically Salmonella typhi, and to elucidate the molecular mechanisms involved in these interactions. The expression of genes involved in TLR signalling and cytokine secretion was analysed.The aim of the present study was to investigate the capacity of L. paracasei CNCM I-4034 were studied in human DCs. The DCs were incubated with the probiotic (live bacteria or CFS) and the pathogen, either individually or together. Our data indicate that probiotic bacteria and their CFS can induce cytokine secretion; this induction was similar in response to probiotic or CFS stimulation. As shown in S. typhi CECT 725) or LPS to DCs markedly increased the secretion of pro-inflammatory cytokines. In response to stimulation with the probiotic or its supernatant and the enteropathogen, the secretion of all of the pro-inflammatory cytokines .TGF-β levels clearly showed that L. paracasei was as effective as the live probiotic bacteria in reducing the secretion of pro-inflammatory cytokines, suggesting that this probiotic secretes metabolites or factors with anti-inflammatory properties.Interestingly, the supernatant of We also evaluated the expression patterns of genes within the TLR signalling pathway. These receptors regulate a variety of different genes, including those that function as transcriptional activators, such as NFκB, whose induction results in the expression of a wide variety of cytokines S. typhi for 4 hours resulted in an upregulation of TLR9  significantly reduced the ability of Salmonella to induce IL-6, IL-8, IL-12p 70 and TNF-α. Interestingly, L. paracasei was not able to induce IL-6 secretion. This effect was observed in the presence or absence of pathogens. This observation contrasts with that of Weiss et al. who reported that lactobacilli and bifidobacteria are potent inducers of IL-6 L. paracasei decreased TNF-α production in response to Salmonella. During the inflammatory process, TNF-α functions at the apex of the inflammatory cascade Salmonella. In addition, Mileti et al. demonstrated that Lactobacillus species can be classified as either immunostimulatory or immunomodulatory. Consistent with this work, L. paracasei CNCM I-4034 and its supernatant appear to be immunomodulatory and could be used to dampen inflammatory responses The co-incubation of human DCs with Salmonella-induced inflammation. Indeed, these data suggest that L. paracasei CNCM I-4034 releases some factors of unknown nature, most likely bacteriocins, because the supernatants did not produce large changes in the profiles of the cytokines released by DCs in the absence of the enteropathogen. Accordingly, it is also important to note that this supernatant is concentrated tenfold, as is any other substance present in the growth medium. We suggest that it is unlikely that this inhibition is caused by growth-derived acidic compounds, such as lactic or acetic acid, because the supernatant used was neutralised to a pH of 7.0. Therefore, the nature of the soluble mediator released by L. paracasei CNCM I-4034 remains to be determined.Interestingly, the culture supernatant by itself could decrease L. paracasei CNCM I-4034 promoted the stimulation of TGF-β2, whereas the supernatant of the probiotic increased the secretion of TGF-β1. TGF-β1 plays important roles in preventing diseases and controlling intestinal homeostasis It is clear that the exposure of DCs to pathogen and probiotic resulted in the release of TGF-β, an anti-inflammatory cytokine. L. paracasei CNCM I-4034 and its CFS stimulated TLR9 expression in the presence or absence of enterobacteria. Similarly, several studies have described increased expression of TLR9 upon the administration of probiotics, such as L. johnsonii and L. caseiTLR2. This TLR recognises peptidoglycan, the main component of Gram-positive bacteria, as Lactobacillus.Gene array analysis is a new approach for evaluating the effects of probiotics on immune cells, and it has provided an overall view of the changes in gene expression patterns in probiotic-treated human DCs L. plantarum BFE 1685 and L. rhamnosus GG, which increase the expression of TLR2 in human epithelial cells L. casei CRL 431 in mice LactobacillusTLR2 and TLR9, activating innate immunity TLR2 stimulation also appears to enhance the epithelial barrier, and it has been shown that the activation of this TLR plays an essential role in resistance to bacterial invasion A similar effect has been described by other authors for Salmonella stimulation. Recently, Villena et al. reported that L.jensenii TL2937 attenuates the inflammatory response triggered by the activation of this TLR in porcine intestinal epithelial cells TLR4 expression was decreased in response of the probiotic and Perhaps most revealing is that the CFS can induce changes in gene expression in the intestine through some factors secreted by the probiotic bacteria. This effect is likely partly responsible for the beneficial effect of probiotics, as highlighted by other authors L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and by the decrease in the production of a broad array of pro-inflammatory cytokines. Our data support the use of a supernatant similar to the one described in this paper as an effective and safe alternative instead of live bacteria. Our results contribute to the understanding of how probiotics exert their immunomodulatory effects.In conclusion, probiotic microorganisms can apparently exert immunoregulatory effects through the secretion of various bacterial compounds that are sensed by host PRRs. This work demonstrates that L. paracasei CNCM I-4034) was isolated from the faeces of breast-fed newborn children. This strain was previously tested for antibiotic tolerance and resistance to gastrointestinal juices, bile salts, NaCl and low pH. The probiotic was grown in Man-Rogosa-Sharpe (MRS) broth medium  at 37°C under anaerobic conditions for 18–24 hours.The probiotic strain  and concentrated tenfold by lyophilization. Supernatants were sterilized by filtering through a 0.22-µm-pore-size filter  and storage at −20°C until use. The supernatant was added 7% v/v.To obtain the CFS, the probiotic strain was grown anaerobically at 37°C in MRS for 24 hours. The supernatant was obtained by centrifugation at 12,000×S. typhi CECT 725. The culture was propagated aerobically in tryptone soy broth .The pathogenic strain was provided by the Spanish Type Culture Collection . The pathogen used in the current study was For the experiments, the pathogen was cultured for 8 hours at 37°C in tryptone soy broth and then subcultured 1∶500 in RPMI 1640 medium  containing 10% foetal bovine serum  at 37°C overnight.+ progenitor cells (haematopoietic stem cells) were supplied by the MatTek Corporation . These cells were seeded in 24-well plates in DC maintenance medium  containing cytokines and antibiotics.DCs generated from umbilical cord blood CD345 DCs/well. For incubations, DC-MM was replaced with RPMI-1640 media. DCs were directly challenged by the addition of the probiotic live bacteria (107 CFU/ml) or CFS, the pathogen (106 CFU/ml) or both. Escherichia coli lipopolysaccharide  at 20 ng/ml was used as a positive control. Negative-control cultures contained unstimulated DCs.Cell cultures were seeded in 24-well plates at a final concentration of 2×102/95% air atmosphere for 4 hours. After incubation, the medium was removed and replaced with fresh DC-MM containing cytokines and antibiotics. After 20 hours, all of the plates were centrifuged at 200×g, and the culture supernatants were collected for cytokine analysis. The cells were collected for RNA extraction. The results shown are the mean ± SEM of three independent experiments.All of the plates were incubated at 37°C in a 5% COIL-1β, IL-6, IL-8, IL-10, IL-12(p40), IL-12(p70), TNF-α, IFNγ, MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5, MDC/CCL22 and TGF-β were measured with MILLIplex™ immunoassays  using the Luminex 200 system according to the manufacturer’s instructions. The results shown are the mean ± SEM of three independent experiments.TLR1, TLR2, TLR3, TLR4, TLR5, TLR9, MYD88, TNF, IRAK-1, IRAK-4, TOLLIP, CASP8, IL-10, TAK-1, JNK, NFKBIA, NFKB-1, TBK-1, MAPK14 and IRF-3. The housekeeping gene GAPDH was used as a control.As previously described, DCs were stimulated with bacteria for 4 hours and collected 20 hours later. The DCs were lysed and total RNA was extracted using the RNAqueous Kit  and treated with Turbo DNase (Ambion) according to the manufacturer’s recommendations. The RNA quality was verified with a Model 2100 Bioanalyser  and the RNA concentration was determined using a RediPlate 96 RiboGreen RNA Quantitation Kit . Real-time RT-PCR analysis of the samples was performed using a Human TLR Signaling Pathway PCR Array , which includes primer pairs specific for 20 genes involved in TLR-mediated signalling pathways: 2 First-Strand Kit (SABiosciences). The cDNA was then subjected to real-time PCR with an RT2 Real-time PCR SYBR Green/ROX Kit (SABiosciences) on an ABI Prism 7500 sequence detector . The PCR conditions were 1 cycle of 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The expression levels of the target genes were normalised to those of untreated DCs (control).Briefly, cDNA was synthesised from total RNA with an RT2 Profiler PCR Array Data Analysis software . The changes in expression or activity levels were expressed as fold changes (Fc). These results reflect the fold increase relative to the control samples (untreated DCs).The expression level of each gene was analysed with RTU-test. The results are presented as the mean + SEM of three independent experiments. All P values<0.05 were considered to be significant and are indicated in the text with an asterisk compared to the controls. In addition, differences between DCs treated with Salmonella and Salmonella and the probiotic/CFS were also evaluated. P values<0.05 were considered to be significant and are indicated with a pound sign (#).NCSS 2007 software  was used for the statistical analysis. The differences in cytokine expression levels and gene expression between treatments were evaluated with the Mann-Whitney


Somatic mosaicism is something that is observed in everyday lives of cytogeneticists. Chromosome instability is one of the leading causes of large-scale genome variation analyzable since the correct human chromosome number was established in 1956. Somatic mosaicism is also a well-known fact to be present in cases with small supernumerary marker chromosomes (sSMC), i.e. karyotypes of 47,+mar/46. In this study, the data available in the literature were collected concerning the frequency mosaicism in different subgroups of patients with sSMC. Of 3124 cases with sSMC 1626 (52%) present with somatic mosaicism. Some groups like patients with Emanuel-, cat-eye- or i(18p)- syndrome only tend rarely to develop mosaicism, while in Pallister-Killian syndrome every patient is mosaic. In general, acrocentric and non-acrocentric derived sSMCs are differently susceptible to mosaicism; non-acrocentric derived ones are hereby the less stable ones. Even though, in the overwhelming majority of the cases, somatic mosaicism does not have any detectable clinical effects, there are rare cases with altered clinical outcomes due to mosaicism. This is extremely important for prenatal genetic counseling. Overall, as mosaicism is something to be considered in at least every second sSMC case, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration, as low level mosaic cases and cryptic mosaics are missed by that. In 1956, the exact chromosomal number in humans was established  and tumoSmall supernumerary marker chromosomes (sSMC) are reported in 0.043% of newborn infants, 0.077% of prenatal cases, 0.433% of mentally retarded patients and 0.171% of subfertile people . They arsSMC are for several reasons still a problem in clinical cytogenetics: (i) they are too small to be characterized for their chromosomal origin by traditional banding techniques and require molecular (cytogenetic) techniques for their identification ; (ii) apHere we focus on the latter mentioned problem – the regularly appearing somatic mosaicism in cases with an sSMC. Mosaicism in association with sSMC is a well-known fact. Crolla (1998)  summariz1). However, there is a strong difference between acrocentric and non-acrocentric derived sSMC: while 72% of acrocentric derived sSMC present no mosaic, 82% of non-acrocentric derived sSMCs are mosaic. According to Liehr (2010)  731/15122, at least 5% of sSMC cases have, after a detailed molecular cytogenetic analysis, a more complex mosaicism than suggested after simple cytogenetic diagnostics. In 20% of these cases, unexpected complex somatic mosaicism was detectable where cytogenetics did not suggest any mosaicism, i.e. in cases 04-U-7, 08-W-p11.2/1-2, 11-O-p11.1/2-1, 11-U-9, 13-U-13, 15-W-q11.1+q11.2/1-1, 21-O-q11.21/1-1, 21-U-5, 22-O-q11.1/5-1, 22-O-q11.1/5-2, 0X-W-p11.?3/1-1, 0X-W-p11.21/1-1 [2). The real grade and complexity of mosaicism seems to be even slightly higher as recently repeatedly cryptic mosaicism was detected in sSMC cases by molecular cytogenetics. There were either cases showing an sSMC in all studied metaphase spreads, however, interphase-FISH in uncultured cells showed a mosaic situation like in case 16-CW-2 . More of1.21/1-1 . InteresCryptic mosaicism appears as some sSMC tend to rearrange and/or be reduced in size during karyotypic evolution. This can lead to double ring formation or inverted duplication starting from a centric minute-shaped chromosome and in the end to the formation of different variants and a highly complex mosaic as some of the new variants can also be degraded in a subset of the studied cells .In summary, somatic mosaics are to be expected in at least 50% of sSMC cases with normal basic karyotype. More complex mosaics can be met in up to 10% of the cases; however, the overall rate of mosaic cases is not significantly altered by cryptic mosaicism, while the genetic complexity of individual cases may be severely influenced.3). While, cases with acrocentric derived sSMC tend to be by mosaic only in 54% of the cases, non-acrocentric derived ones are always mosaic with an exception of 1/29 reported patients (Table 3). Thus, in sSMC duplication cases we find a similar situation as in cases with one single sSMC and a karyotype 47, +mar concerning mosaicism. sSMC in a small subset of cases tend to duplicate, leading to a karyotype 48,+marx2 . Up to nMultiple sSMC cases  differ fSomatic mosaicism is reported to different extents in four sSMC-related syndromes.Patients suffering from Pallister-Killian-syndrome (PKS) due to the presence of an additional isochromosome 12p are known to have somatic mosaicism in practically every case. In peripheral blood the +(12p) tends to be lost either already during pregnancy or shortly after birth in practically all cells. In the alternatively studied skin fibroblasts, the sSMC derived from chromosome 12 is normally easily to detect in >70% to 100% of the cells . HoweverIn isochromosome 18p syndrome mosaicism is rather rare. But also here exceptional cases are known having the full clinical phenotype but normal karyotype in some of the body cells . In casTo the best of our knowledge no mosaic cases are known by now for Emanuel-syndrome (ES) . Also inOverall, somatic mosaicism is, compared to other sSMC derived from the corresponding chromosomes, over-represented in PKS (100% vs. 79%) and under-represented in i(18p) syndrome (4% vs. 67%), ES (0% vs. 32%) and CES (3% vs. 32%).26 sSMC cases with Prader-Willi-syndrome (PWS) and 7 with Angelman-syndrome (AS) can be found in the literature . 15 of tAnother rare cytogenetic variant of sSMC presence is that of a structurally abnormal but balanced karyotype (McClintock mechanism) . Such caFor mosaicism in neocentric sSMC formed by McClintock mechanism,  the same4). Strikingly, as in centric sSMC, mosaicism is more frequent in non-acrocentric derived compared to acrocentric derived ones (58% vs. 24%). In general, in at least around 50% of the cases with a neocentric sSMC somatic mosaicism is observable  and one not (09-U5) . For triFor sSMC, at present additionally to a trisomy 21 (Down-syndrome), information on mosaic status is available in 16 cases; 7 of those 44%) have somatic mosaicism with a cell line 47, +21 without sSMC [% have soOverall, mosaicism is a frequent finding when an sSMC is present additionally to a numerically abnormal basic karyotype. Summarizing all above mentioned groups, 1626 of 3124 cases with sSMC 52%) present with somatic mosaicism. Even though, expressed to a different extent in various sub-groups, mosaicism is something to be considered in at least every second such case. However, if a specific genetic imbalance caused by an sSMC is known to be harmful, in the overwhelming majority of the cases there is no influence of the grade of somatic mosaicism detectable in peripheral blood or amnion cells and the observed clinical effects. This seems to be due to the fact that the mosaicism rate in different human tissues is practically not predictable and very variable % present. Even thKnowing that somatic mosaicism happens in ~50% of the cases with sSMC, array-CGH studies cannot be offered as a screening test to reliably detect this kind of chromosomal aberration. On the one hand, low level mosaic cases and on the other hand, cryptic mosaics are missed. Thus, cytogenetics is still the gold-standard to detect any kind of chromosomal aberration, which then, in further steps can be characterized by molecular (cyto-) genetic approaches. Interestingly, acrocentric and non-acrocentric derived sSMC are differently susceptible to mosaicism; acrocentric derived ones are hereby the more stable ones. This holds true for centric and neocentric sSMC, and an explanation is therefore at present not available. Somatic mosaicism is a feature of the human body, which has to be considered much more than up to now in future. It is known as a fact, but not understood why man with age  develops something like a ‘Turner-syndrome-mosaic’ 46,XY/45,X. Similarly, in cases with sSMC it is known since years, that PKS patients lose the i(12p) in peripheral blood or that some inv dup(15) sSMC are stable and cytogenetically identical ones in another carrier are not. For all these facts to the best of our knowledge, no studies were undertaken to come closer to an understanding of these phenomena. Here we present, some kind of starting point for such studies, for the first time a detailed ‘mosaicism map’ for the different subtypes of sSMC.


Tumor models were obtained by transplantation of PC-3M tumor cells into male BALB/c nude mice. Animals were randomly divided into control, 32P-chromic phosphate (32P-CP) colloid and 32P-CP-PLLA groups . A series of indices were investigated, including apoptosis of tumor cells, rate of apoptosis, expression of caspase 3 and 8, biodistribution and intratumoral concentration of 32P-CP-PLLA, intensity of radioactivity, tumor volume and microvessel density (MVD). Highly concentrated radioactivity of 32P-CP-PLLA in the tumor mass was detected by single photon emission computed tomography (SPECT) scanning. The residual activities of the 32P-CP-PLLA and 32P-CP colloid groups were 3.02±0.32 and 1.76±0.31 MBq, respectively, on day 14 following treatment. The tumor inhibition rates were 67.24±3.55 and 55.92±7.65%, respectively (P<0.01). Necrotic changes, in conjunction with apoptosis, were observed in the treatment group. MVD values for the 32P-CP-PLLA and 32P-CP colloid groups were 28.24±10.07 and 36.15±11.06, respectively. 32P-CP-PLLA showed an excellent capacity for killing tumor cells, inducing apoptosis and inhibiting angiogenesis.The aim of the present study was to investigate the antitumor effects and tissue distribution of  Prostate cancer is ranked second among cancer-related mortalities in American male citizens . Radioth125I, is the preferred treatment for low risk prostate cancer in a number of countries and the outcome for moderate- and high-risk prostate cancer patients remains satisfactory  colloid has been applied for the treatment of intracavitary cancer  control of the distribution of microspheres or colloid outside the tumor mass; and iii) reduction or even elimination of toxicity and side effects. In addition, the complexities associated with dose calculation and clinical practice prevent the development of 32P-CP colloid for clinical use. Thus, the identification of a novel vector for the transportation of 32P radionuclide is crucial for low dose rate brachytherapy.y cancer  and its y cancer . Howeverd tumors –17, howe32P-CP-PLLA microparticles were produced with these characteristics and degraded continuously under specific temperatures and humidities. In addition, a comparative study was performed in nude mice with human prostate cancer to investigate the differences in the pharmacokinetic profile and treatment efficiency in 32P-CP colloid and 32P-CP-PLLA groups.Poly (L-lactic acid) (PLLA) has been widely used as a drug delivery system due to its excellent biocompatibility and biodegradability –20. It i2 in a humidified atmosphere, using Roswell Park Memorial Institute medium supplemented with 10% heat-inactivated fetal bovine serum  and L-glutamine. A total of 90 healthy male BALB/c nude mice  at 4–6 weeks old and 18–22 g were maintained in Streamline® cabinets  at 25–27ºC and a humidity of 40–50%.PC-3M human prostate cancer cells  were maintained in stationary monolayer cultures at 37ºC, with 5% CO32P-CP-PLLA was prepared as described previously  and dehydrated alcohol was used as a dispersant. The affinity between PLLA and the colloid was modulated by surface-active agents. The mixture was treated by ultrasonication for 30 min, kept at room temperature for 1 h and dried in the drying vacuum oven at 60ºC. Pentobarbital  was injected via peritoneal injection following anesthesia. The paracentesis needle was inserted into the center of the tumor along the long axis, followed by injection with microparticles. The radioactivity concentration was 0.39 GBq/ml (10.5 mCi/ml) and the needle was removed once resistance was felt. Following dilution with a physiological solution of sodium chloride, the intratumoral injection activity was 7.4 MBq (0.05 ml) for the colloid.eviously . Briefly6 PC-3M cells on the right upper flank was performed to induce tumorigenesis. Continuous measurements of tumor dimensions were conducted using a caliper. Tumor volume was calculated by the following formula: Tumor volume = length/2 × width2.A subcutaneous inoculation of 2×1032P-CP colloid , 32P-CP-PLLA  and control  groups when tumor volume reached 8 mm in diameter. All animal experiments were carried out according to national laws. This study was approved by the ethics committee of Changzhou No. 3 People’s Hospital.Animals were randomly divided into 32P-CP-PLLA and 32P-CP colloid distribution was investigated using SPECT fitted with a low-energy, general-purpose collimator . The single pinhole SPECT system was operated in a routine manner. In brief, a cylinder with a diameter of 25 mm, designed to be tight fitting for mice, was positioned directly and horizontally above the pinhole aperture. A mechanical support allowed for the precise and manual adjustment of the cylinder in two directions; the distance of the cylinder to the pinhole aperture, which equals the radius of rotation, and along the axis of the cylinder to select the field of view. The pinhole collimator was connected to an ADAC ARC 3000 scintillation camera  and had a focal length of 320 mm and an opening angle of 60º. The energy window was set at 78 keV, 30% width and 30 min harvest time. SPECT images were captured at 1 and 12 h and 1, 2, 4 and 8 days post-administration.Bremsstrahlung scintigraphy of Tumor volume was determined every two days following treatment. The tumor inhibition rate was calculated on day 14 using the following formula: tumor inhibition rate = (W1 – W2)/W1 × 100; where W1 and W2 represent the average weight of tumor volume in the control and treatment groups, respectively. For the calculation of the tumor inhibition rate, 5 mice were sacrificed in each group for conventional histological examination by formalin fixation and paraffin embedding. Hematoxylin and eosin staining was performed for the monitoring of sections.Apoptosis was measured using a TUNEL assay kit  at 1, 6, 12, 24 and 72 h  following treatment. The procedures were performed according to the manufacturer’s instructions. The apoptosis index was calculated as the ratio of apoptotic cells to total cells.Expression of caspase 3 and 8 was evaluated using caspase 3 and caspase 8 Activity Assay kits according to the manufacturer’s instructions . The Bio-Rad Microplate 550 Reader  was used to monitor the absorbance at 405 nm. The activities of caspase 3 and 8 were evaluated by the ratio of the optical density of the inducer to the negative control.Slides stained with anti-CD34 monoclonal antibody were examined via microscope. The cells that were positive for CD34 were designated as a vessel and initially observed at ×100 magnification to select the high density areas of microvessels. Next, the microvessel density (MVD) of the CD34-positive cells was investigated at ×200 magnification.A β-radioactivity meter was used to determine radioactivity and final radioactivity was calculated as described previously .All data are presented as mean ± standard deviation. Statistical analyses were performed using SPSS 13.0 Software  and statistical significance was detected by ANOVA. P<0.05 was considered to indicate a statistically significant difference.32P-CP colloid group, irregular and eccentric growth of the tumor mass and local recurrence was identified in 1 mouse. No eccentric growth or recurrence was observed in the 32P-CP-PLLA group.Anorexia was identified in the control group and skin ulcers were detected in the right lower extremities of 2 mice. However, no anorexia or skin ulcers were observed in the treatment groups and tumor growth was reduced significantly compared with the control group. In the 32P-CP-PLLA microparticles, biodistribution was detected by SPECT imaging. During the initial stages, microparticles were concentrated at the implantation sites, however, over time, a low radioactive uptake (RAU) of 32P-CP-PLLA was identified in the tumor mass compared with the baseline levels. In addition, no shifting or loss of 32P-CP-PLLA was identified  with MVD values of 60.71±8.21, 36.15±11.06 and 28.24±10.07 for the control, colloid and microparticle groups, respectively .32P has been hypothesized to represent the ideal radionuclide for brachytherapy. In the present study, PLLA was used as the delivery vector for 32P-CP to modulate the target orientation and safety of internal radiation therapy. PLLA has excellent biodegradation and biocompatibility and was approved as a pharmaceutical adjuvant by the FDA in 1997. PLLA is now widely used as a drug delivery system  was identified between the 32P-CP colloid and 32P-CP-PLLA groups, therefore, indicating that 32P-CP-PLLA is likely to maintain its concentration in the tumor mass using the PLLA delivery system.It has been previously identified that cell proliferation and apoptosis are markedly associated with prostate cancer. In the present study, necrosis and apoptosis were identified in the tumor mass of the treatment groups, indicating that 32P. Lower MVD values were identified in the 32P-CP-PLLA group compared with the colloid group indicating that the microparticles may inhibit angiogenesis by improving the radioactive dosage in the tumor mass. Gellman et al reported that intimal proliferation is likely to be induced by a lower dose of internal irradiation . This is likely to be associated with the temporarily high local dose in the 32P-CP colloid group during the early stages (~6–12h) compared with the gradual degradation of 32P-CP-PLLA.Two major pathways that have been identified as associated with apoptosis are the extrinsic and intrinsic pathways . The extrinsic pathway is activated by ligand-activated death receptors, including Fas ligand (FasL). Caspase 8 functions as a significant initiation factor for apoptosis, with caspase 3 as the concluding indicator for apoptosis and the core molecule of the Fas/FasL signaling pathway . The res32P-CP colloid and 32P-CP-PLLA may kill tumor cells, induce apoptosis and inhibit angiogenesis. However, the tumor volume was reduced in the 32P-CP-PLLA group compared with the 32P-CP colloid group at 8 days post-treatment, and the most significant difference was identified on day 14. This demonstrated an enhanced retention time of 32P-CP-PLLA at the target site, and an accumulated yield of radiation was identified in the 32P-CP-PLLA group following the gradual delivery of the radioactive source of equivalent radioactivity levels. Therefore, this indicated that 32P-CP-PLLA functions via low dose and gradual delivery of the radioactive source.The present results showed that 32P-CP-PLLA had the following distinct characteristics: i) enhanced metabolism of the drug at the target site and reduced dose distribution outside the tumor mass by degradation and delayed release of the drugs; ii) permanent stagnation and complications were avoided due to an excellent degradation capacity of the transplanted microparticles, under specific conditions; iii) biological metabolism of 32P-CP-PLLA was regulated by 32P-CP-PLLA activity in vivo with specific dosages convenient for individual therapy; and iv) as a polymer, modification of the structure of 32P-CP-PLLA was applicable and may aid the possible development of a delivery system with increased efficiency, including the synergic sensitization of chemotherapeutics.Overall, the results demonstrated that 32P-CP-PLLA microspheres in the clinical treatment of prostate cancer, the microspheres may be precisely implanted into prostate cancer lesions under the three-dimensional radiotherapy planning system. In addition, as the microspheres are likely to be degraded gradually over a specific period, repetitive implantation is possible to prevent the lymph node metastases and micrometastasis of prostate cancer. Therefore, 32P-CP-PLLA may decrease the recurrence of prostate cancer and improve the survival rates of patients. In addition, the range of β-array radiation emitted by 32P was ~4 μm and thus it may cause slight or no damage to sexual function and decrease the occurrence of urinary incontinence, urethral stenosis and rectal complications. It is therefore hypothesized that 32P-CP-PLLA may represent an innovative method for the treatment of prostate cancer.With regard to the application of


Momordica charantia (bitter gourd) seed extracts. Fasting blood glucose levels were evaluated before and after administration of different fractions of the seed extract. Among the three fractions tested, fraction Mc-3 (15 mg/kg b.wt.) showed the maximum anti-hyperglycemic activity and reduced blood glucose levels in experimental diabetic rats significantly. The activities of the key regulatory enzymes of glucose metabolism  were determined in Mc-3-treated diabetic animals. Once-daily administration of the fraction Mc-3 for prolonged period of 18 days to the experimental diabetic animals did not result in any nephrotoxicity or hepatotoxicity as evident from insignificant changes in biochemical parameters indicative of liver and kidney functions. Further fractionation of the fraction Mc-3 by size exclusion chromatography resulted in a fraction, designated Mc-3.2, possessing anti-hyperglycemic activity. The fraction Mc-3.2 showed the presence of a predominant protein band of ~11 kDa on SDS-PAGE. Loss in anti-hyperglycemic activity of the Mc-3.2 upon protease treatment indicates the proteinaceous nature of the anti-hyperglycemic principles. Overall, the results suggest that Momordica charantia seeds contain an effective anti-hyperglycemic protein(s) which may find application in treatment of diabetes without evident toxic effects.The present study evaluates anti-hyperglycemic activity of fractionated  Diabetes mellitus, a metabolic disorder, is a major global health concern with a projected rise in prevalence from 171 million in 2000 to 366 million in 2030 [Momordica charantia (Mc), commonly known as bitter gourd, is one of the most used plants for the treatment of diabetes and related conditions in traditional system of medicine world over [rld over –6. The erld over –10, or grld over , 12. In rld over , anti-hurld over , anti-ulrld over  and hyporld over . While a number of reports have been put forth demonstrating the anti-hyperglycemic activity of Mc, systematic studies to evaluate the effect of prolonged treatment with the fractionated seed extracts on the blood glucose levels together with acute toxicity studies have not been carried out.Therefore, the present study focuses on the bioassay-guided fractionation of the acid-ethanolic extract of Mc seeds to identify the fraction that contains the active principle(s) responsible for the anti-diabetic activity. Further, the effect of long-term treatment with the active fraction on glycemic control and on the liver and kidney functions in diabetic rats was also investigated, as these studies would aid in evaluating the hepatotoxic and nephrotoxic effects, if any, of the prolonged treatment and proving to be nontoxic will facilitate the use of the active fraction(s) for diabetes management.Momordica charantia seeds (“Pusa Vishesh” variety) were procured from Indian Agriculture Research Institute (IARI), Pusa, New Delhi [ew Delhi  in largeRandom bred male Wistar rats (12–14 weeks) were housed in the Small Animal Facility of the Jawaharlal Nehru University. The animals were provided with rat feed  and water ad libitum. The use of animals was duly approved by the Institutional Animal Ethics Committee of the JNU, and the guidelines prescribed by the Institutional Animal Ethics Committee, JNU, New Delhi, were followed while handling animals. Extraction of seeds was done essentially as described earlier  with minThe male Wistar rats were made diabetic by using alloxan. Briefly, alloxan was administered i.p. after starving the animals for 36 hrs at a dose of 150 mg/kg body weight (b.wt.). Animals were stabilized for three days by insulin administration, 1-2 units per day for 2 days. Only those animals having blood glucose level more than 300 mg per 100 mL blood were selected for further analysis. The diabetic animals were grouped into experimental groups each containing minimum 5 rats. The doses of different fractions are expressed in terms of their protein content. Different groups were treated with different Mc seed fractions (15 mg/kg b.wt.). Diabetic animals treated with saline (vehicle) were included in the study as negative control. A group of diabetic animals treated with protamine zinc insulin  served as standard reference control. A group of normal untreated non-diabetic animals was also included in the study. Fasting serum glucose (6 h fasting) was measured in blood drawn from the tail vein during the study period using glucose oxidase-peroxidase method  or a gluIn order to determine the time by which the active fraction is able to bring about anti-hyperglycemic effect, a short-term (0–4.5 h) study was conducted by measuring the blood glucose levels within the indicated periods after administration. Short-term time kinetics of the active fraction in diabetic rats was determined with a dose of 15 mg/kg b.wt. administered intraperitoneally (i.p.) or insulin (10 IU/kg b.wt). The blood glucose levels were measured at different time intervals. Optimum dose of the active fraction was determined by administering the animals with different concentrations (5–25 mg/kg b.wt.) of the active fraction. Blood glucose levels were measured at 3 h after administration. The rats were divided into different groups (five rats in each group): Group I—PBS-treated normal non-diabetic controls, Group II—PBS-treated diabetic rats, Group III—diabetic rats treated with 15 mg/kg b.wt. of the active fraction, and Group IV—the diabetic rats treated with protamine zinc insulin (10 IU/kg b.wt.). The first two groups of rats were given saline (vehicle) daily. The extract and insulin were administered at the selected dosage to Groups III and IV, respectively, every day for 18 days. Body weights of the untreated and treated animals were monitored throughout the study period. The rats were bled prior to sacrifice on the last day of the treatment by cervical dislocation. Serum was collected and subjected to biochemical analysis for hepatic function markers using assay kits from AutoZyme, India, by the method of Penttila et al. , and ren4HCO3 (pH 7.2–7.4). The collected fractions were analyzed by SDS-PAGE and silver staining. The peak fractions designated as Mc-3.1, Mc-3.2, and Mc-3.3 were analyzed for anti-hyperglycemic activity using experimental diabetic rats as described before. The enriched fraction showing maximum biological activity was further fractionated by gel filtration chromatography using Sephacryl S-100 HR in 0.2 M NHThe most active fraction obtained after gel filtration chromatography was assessed for its ability to induce glucose tolerance in normal Wistar rats. The rats were administered with 2 g/kg of glucose (i.p.) after 30 min of the test fraction at a dose of 15 mg/kg b.wt. (i.p.). Serum glucose levels were measured in the blood samples collected from the tail vein prior to glucose administration (considered as 0 h) and at different time intervals after glucose administration. t-test for paired data of different levels of significance. All the results were expressed as mean ± S.D. P values less than 0.05 were considered significant.All the results were analyzed statistically using one-way ANOVA or Student's paired The crude acid ethanolic extract upon centrifugation gave rise to fractions Mc-0 (insoluble fraction) and Mc-1. Since fraction Mc-0 contained insoluble material, it was not administered in the animals. The fraction Mc-1 and the fractions Mc-2 and Mc-3 derived from Mc-1 were tested for their anti-hyperglycemic potential in experimental diabetic rats. As evident, fraction Mc-1 at the tested dose (15 mg/kg b.wt.) was able to lower blood glucose levels . After hSince fraction Mc-3 was found to possess enriched anti-hyperglycemic activity, it was desirable to study how quickly fraction Mc-3 could exert its anti-hyperglycemic effect after administration. Therefore, blood glucose levels of the Mc-3 treated animals were measured at different time after administration for a short period of 4.5 h. As shown in In order to assess the optimum concentration of Mc-3 that was able to bring about significant reduction in serum glucose levels of diabetic animals, the animals were administered with different concentrations of the fraction Mc-3 (5–25 mg/kg b.wt.) and the blood glucose levels were determined at 3 hr after administration . It was observed that Mc-3 showed an increased reduction in blood glucose levels till 15 mg/kg b.wt. No further reduction in the blood glucose levels was observed when the animals were treated with a higher concentration (20 mg/kg b.wt.) of fraction Mc-3 . In order to assess the long-term effect of fraction Mc-3, the diabetic animals were maintained on fraction Mc-3 for a period of 18 days in order to assess if the continued administration of the active fraction Mc-3 had some toxic or undesirable effect on the liver and kidney functions. As expected, the blood glucose levels of the animals treated daily with Mc-3 were lower when compared to initial levels prior to the treatment . A singlM. charantia seed extracts. The Mc-3 treated animals had a 100% survival during the study period of three weeks with a normal behaviour, whereas the untreated control diabetic animals were lethargic, week and showed only about 60% survival by the end of three-week period. After treatment, the serum activities of hepatic function markers: serum glutamic pyruvic transaminase (SGOT), serum glutamic oxaloacetic transaminase (SGPT), gamma-glutamyl transpeptidase (GGT), and the levels of renal function markers (urea and creatinine) were measured. Daily administration of insulin for 18 days resulted in significantly reduced levels of SGOT, SGPT, and GGT in comparison to PBS-treated diabetic controls. Similarly, the Mc-3-treatment of the diabetic animals for a period of 18 days also resulted in a significant reduction in the serum glutamic pyruvic transaminase (SGOT), serum glutamic oxaloacetic transaminase (SGPT), and gamma-glutamyl transpeptidase (GGT) when compared to PBS-treated control diabetic animals . LikewisIn order to determine if the reduction in serum glucose levels by Mc-3 treatment is due to increased glucose utilization, liver and muscle glycogen levels were measured. A significant reduction in both the liver and muscle glycogen levels was observed in the diabetic animals when compared to normal controls. Mc-3 treatment resulted in an increase of ~63% and ~80% in glycogen content of liver and muscle when compared to the PBS-treated diabetic animals . Both thThe effect of Mc-3 treatment on key enzymes of glucose utilization in the livers of treated animals was also evaluated. To understand the possible mechanism of action, the diabetic animals were administered with either fraction Mc-3 or same volume of saline and maintained for 3 days on respective treatment. On day 4, the animals were injected with the test samples and were sacrificed after 3 hr of injection. Activities of the various regulatory enzymes were estimated in the livers of the control  and Mc-3-treated diabetic animals .Control diabetic animals showed reduced levels of all the enzymes when compared to normal controls. It was observed that fraction Mc-3 resulted in a significant increase (~2 fold) both in pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PDH) activities, whereas only ~1.3 and ~1.6 fold increase in the specific activity of hexokinase (HK) and malic enzyme (ME) was noted. However, no significant change in lactate dehydrogenase (LDH) activity was observed. Insulin-treatment also resulted in an increase in the activities of all the enzymes and restored their levels to that observed in normal non-diabetic animals. Earlier reports have indicated that the Mc fruit/seeds contain polypeptides that are capable of reducing blood glucose levels , 29. LosM. charantia seeds was proteinaceous in nature. SDS-PAGE analysis of the active peak fraction Mc-3.2 showed that this consisted of dominant protein(s) of ~10–12 kDa on a non-reducing SDS-PAGE , Mc-3.2 (fractions 35–40), Mc-3.3 (fractions 43–46), Mc-3.4 (fractions 47–49), and Mc-3.5 (fractions 50–53). Fraction Mc-3.1 could not be evaluated for its anti-hyperglycemic activity due to small amounts. Of the remaining fractions, only fraction Mc-3.2 resulted in significant decrease (~40%) in blood glucose levels . The othSDS-PAGE . To check the ability of fraction Mc-3.2 to maintain normoglycemia in normal animals, glucose tolerance test was performed. Administration of Mc-3.2 resulted in faster clearance of blood glucose levels in normal rats when compared to PBS-treated controls, without causing hypoglycemia . At thisHyperglycemia associated with diabetes mellitus can be controlled by diet management, exercise, oral hypoglycemic agents, and insulin therapy. Both insulin therapy and oral hypoglycemic agents have their own side effects and adaptation problem. The secondary complications of diabetes that appear with lapse of time are actually the major cause of morbidity and mortality. Therefore, development of new approaches for treatment of diabetes that can reduce blood sugar level with better adaptation is desirable.Dioscorea dumetorum extracts has been reported [The evaluation of plants and especially of their active principles is a logical way of searching for new drugs to treat the disease. However, the presence of undesirable hyperglycemic substances along with the hypoglycemic components in reported . Thus, tMomordica charantia has been used in indigenous system of medicine since a long time. Several reports have been put forth suggesting different parts of the plant to possess hypoglycemic activity. These have been found to be effective in chemically  induced experimental diabetic rats [tic rats –10.Momordica charantia [M. charantia referred to as Charantin and p-insulin have been reported to lower fasting blood sugar (~25–40%) in experimental diabetic animals [M. charantia seeds has resulted in ~40% reduction in blood glucose levels within 3 h of treatment with an onset of reduction within 1 h of treatment. The anti-hyperglycemic effect brought about by the Mc-3 fraction of M. charantia seeds was comparable to that observed with insulin treatment of the diabetic animals. Our observations in the insulin-treated animals are in agreement with the earlier reports where in a sharp decrease in the fasting serum glucose levels was observed in protamine zinc insulin within two hours [Many small molecular weight proteins, polypeptides with hypoglycemic and/or anti-hyperglycemic activities have been isolated from harantia –33. Othe animals , 34. In wo hours . Like eawo hours , 37.The anti-hyperglycemic activity of the fraction Mc-3 at a much lower dose (15 mg/kg b.wt.) is significantly higher than that observed with acid-ethanol extract (fraction Mc-1) or other fraction Mc-2 derived from Mc-1. It appears that fraction Mc-2 consists of some toxic compounds that caused skin lesion and necrosis at the site of injection. Over a period of 18 days treatment daily, the blood glucose levels of fraction Mc-3-treated diabetic rats were significantly reduced, a desirable criterion for any potential anti-diabetic agent. Also, no hypoglycemic condition was observed in the treated animals. The fraction Mc-3 was effective at much lower concentration (15 mg/kg b.wt.) when compared to that of the crude ethanolic extracts of other plants which were found to be effective in the range of 100–500 mg/kg b.wt. in diabetic rats –40. ThusS. cordatum extract, capable of bringing down blood glucose levels [Hyperglycemic condition due to partial or total lack of insulin arises because of disturbances in glucose metabolism caused by a decrease in several key enzymes of glycolysis, namely, glucokinase, phosphofructokinase, and pyruvate kinase, thus resulting in impaired peripheral glucose utilization and augmented hepatic glucose production . Also, ce levels . Also, aIt is of interest to note that the treatment of diabetic animals with Mc-3 resulted in reduction in the serum levels of marker enzymes of liver function, namely, SGOT, SGPT, and GGT. An increase in the serum levels of hepatic function marker enzymes generally arises due to necrotized liver conditions caused by chronic diabetes , 47. FurM. charantia have employed extracts of whole fruit, pulp, and leaves in experimental diabetic animals of glucose-loaded rats [M. charantia seeds. The anti-hyperglycemic principle(s) is highly effective in bringing down blood glucose levels to near normoglycemia, at much lower concentrations without causing any adverse effect on liver and kidney function of the treated animals. Studies are in progress to further purify and characterize the active proteinaceous principle present in fraction Mc-3.2 of M. charantia, which will be helpful in understanding the molecular mechanisms by which anti-hyperglycemic effect is brought about.Earlier reports on the anti-diabetic potential of ded rats –12. The